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• W2029303304 abstract "The widely distributed protein-l-isoaspartate(d-aspartate)O-methyltransferase (PIMT; EC 2.1.1.77) is postulated to play a role in the repair or metabolism of damaged cellular proteins containing l-isoaspartyl residues derived primarily from the spontaneous deamidation of protein asparaginyl residues. To evaluate the functional consequence of PIMT-catalyzed methylation on the stability of isoaspartyl-containing proteins in cells,Xenopus laevis oocytes were microinjected with both deamidated and nondeamidated forms of recombinant chicken calmodulin (CaM) containing a hemagglutinin (HA) epitope at its N terminus. Processing of HA-CaM was monitored by electrophoretic analysis and Western blotting of oocyte extracts. The experiments indicate that deamidated HA-CaM is degraded after microinjection, while nondeamidated HA-CaM is stable. Kinetic analysis is consistent with the entry of microinjected HA-CaM into two intracellular pools with distinct hydrolytic stabilities. The larger, more stable pool may consist of HA-CaM bound to the heterogeneous pool of oocyte CaM binding proteins detected by an overlay procedure. Enzymatic methylation of deamidated HA-CaM with purified PIMT prior to injection results in its stabilization. Conversely, inhibition of endogenous oocyte PIMT with sinefungin, a nonhydrolyzable analog of S-adenosylhomocysteine, increases the rate of deamidated HA-CaM degradation. These results are consistent with a role for PIMT-catalyzed methylation in the repair of damaged cellular proteins. The widely distributed protein-l-isoaspartate(d-aspartate)O-methyltransferase (PIMT; EC 2.1.1.77) is postulated to play a role in the repair or metabolism of damaged cellular proteins containing l-isoaspartyl residues derived primarily from the spontaneous deamidation of protein asparaginyl residues. To evaluate the functional consequence of PIMT-catalyzed methylation on the stability of isoaspartyl-containing proteins in cells,Xenopus laevis oocytes were microinjected with both deamidated and nondeamidated forms of recombinant chicken calmodulin (CaM) containing a hemagglutinin (HA) epitope at its N terminus. Processing of HA-CaM was monitored by electrophoretic analysis and Western blotting of oocyte extracts. The experiments indicate that deamidated HA-CaM is degraded after microinjection, while nondeamidated HA-CaM is stable. Kinetic analysis is consistent with the entry of microinjected HA-CaM into two intracellular pools with distinct hydrolytic stabilities. The larger, more stable pool may consist of HA-CaM bound to the heterogeneous pool of oocyte CaM binding proteins detected by an overlay procedure. Enzymatic methylation of deamidated HA-CaM with purified PIMT prior to injection results in its stabilization. Conversely, inhibition of endogenous oocyte PIMT with sinefungin, a nonhydrolyzable analog of S-adenosylhomocysteine, increases the rate of deamidated HA-CaM degradation. These results are consistent with a role for PIMT-catalyzed methylation in the repair of damaged cellular proteins. protein-l-isoaspartate(d-aspartate)O-methyltransferase calmodulin hemagglutinin hemagglutinin-tagged calmodulin S-adenosylmethionine 2-(N-morpholino)ethanesulfonic acid matrix assisted laser desorption-time of flight adrenocorticotropic hormone polyacrylamide gel electrophoresis. All living cells contain enzymatic systems that maintain a functional pool of cellular proteins by catalyzing the refolding, repair, or removal of structurally damaged proteins (1Stadtman E.R. Science. 1992; 257: 1220-1224Crossref PubMed Scopus (2396) Google Scholar, 2Gething M.-J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3601) Google Scholar). In some cases, these enzymes recognize modified or abnormal amino acid residues that have arisen spontaneously during the aging of the protein (3Clarke S. Annu. Rev. Biochem. 1985; 54: 479-506Crossref PubMed Google Scholar, 4Aswad D.W. Curr. Opin. Cell Biol. 1989; 1: 1182-1187Crossref PubMed Scopus (13) Google Scholar). One such activity is a protein protein-l-isoaspartate(d-aspartate)O-methyltransferase (PIMT; EC2.1.1.77)1 that appears to be of ancient origin, with homologues identified in bacterial, plant, and animal cells (5Johnson B.A. Ngo S.Q. Aswad D.W. Biochem. Int. 1991; 24: 841-847PubMed Google Scholar, 6Kagan R.M. McFadden H.J. McFadden P.N. O'Connor C. Clarke S. Comp. Biochem. Physiol. 1997; 117B: 379-385Crossref Scopus (44) Google Scholar). All of the widely distributed PIMT activities specifically recognize l-isoaspartyl residues that can spontaneously arise from either the deamidation of protein asparaginyl residues or the isomerization of protein aspartyl residues (7Geiger T. Clarke S. J. Biol. Chem. 1987; 262: 785-794Abstract Full Text PDF PubMed Google Scholar, 8Stephenson R.C. Clarke S. J. Biol. Chem. 1989; 264: 6164-6170Abstract Full Text PDF PubMed Google Scholar). It has been demonstrated for several proteins, including epidermal growth factor, calmodulin (CaM), and calbindin (9Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar, 10George-Nascimento C. Lowenson J. Borissenko M. Calderón M. Medina-Selby A. Kuo J. Clarke S. Randolph A. Biochemistry. 1990; 29: 9584-9591Crossref PubMed Scopus (35) Google Scholar, 11Chazin W.J. Kördel J. Thulin E. Hofmann T. Drakenberg T. Forsén S. Biochemistry. 1989; 28: 8646-8653Crossref PubMed Scopus (87) Google Scholar), that the appearance of an isoaspartyl site is correlated with a loss of normal enzyme function. It has been proposed that PIMT-catalyzed carboxyl methylation of isoaspartyl sites is the first step in either the repair or degradation of the damaged protein substrate (3Clarke S. Annu. Rev. Biochem. 1985; 54: 479-506Crossref PubMed Google Scholar, 9Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar, 12Brennan T.V. Anderson J.W. Jia Z. Waygood E.B. Clarke S. J. Biol. Chem. 1994; 269: 24586-24595Abstract Full Text PDF PubMed Google Scholar). Evidence supporting a repair function has come from studies of PIMT-catalyzed methylation in vitro. In several experiments, isoaspartyl-containing synthetic peptides were nearly stoichiometrically converted to the corresponding aspartyl-containing form following carboxyl methylation by PIMT and the ensuing internal rearrangements associated with hydrolysis of the ester, involving a succinimide intermediate (13McFadden P.N. Clarke S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2595-2599Crossref PubMed Scopus (168) Google Scholar, 14Johnson B.A. Murray Jr., E.D. Clarke S. Glass D.B. Aswad D.W. J. Biol. Chem. 1987; 262: 5622-5629Abstract Full Text PDF PubMed Google Scholar). The repair process, however, lacked the strict stereochemical specificity and efficiency characteristic of enzymatic systems, requiring multiple rounds of methylation and demethylation to achieve full repair. Experimental evidence has also been presented implicating PIMT in the repair of damaged protein substrates. When deamidated, isoaspartyl-containing forms of the bacterial HPr phosphocarrier protein (12Brennan T.V. Anderson J.W. Jia Z. Waygood E.B. Clarke S. J. Biol. Chem. 1994; 269: 24586-24595Abstract Full Text PDF PubMed Google Scholar) or calmodulin (9Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar) were used as substrates for the PIMT, carboxyl methylation partially restored enzymatic activity that had been lost as a consequence of the original deamidation. Significantly, however, full enzymatic activity was not recovered, and isoaspartyl residues were not replaced by the original asparaginyl residues. At the present time, there is no evidence supporting the other proposed role for the PIMT in marking damaged substrates for degradation, although proteolytic destruction of damaged proteins would also serve to maintain a functional pool of cellular proteins. Based on the available evidence, which is all derived from experiments using purified systems (9Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar, 13McFadden P.N. Clarke S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2595-2599Crossref PubMed Scopus (168) Google Scholar, 14Johnson B.A. Murray Jr., E.D. Clarke S. Glass D.B. Aswad D.W. J. Biol. Chem. 1987; 262: 5622-5629Abstract Full Text PDF PubMed Google Scholar), it would be premature to exclude a physiological role for the PIMT in the destruction of damaged isoaspartyl-containing proteins. Purified systems may lack hitherto unidentified activities that participate in the processing of isoaspartyl-containing proteins within cells. From these studies, it has become clear that elucidation of the biochemical pathway initiated by carboxyl methylation would be assisted by the development of a model for studying the processing of methylated proteins in intact cells. We have chosen Xenopus oocytes for a model because these cells possess a functional excess of PIMT similar to those activities previously studied in more detail in mammalian systems (15O'Connor C.M. J. Biol. Chem. 1987; 262: 10398-10403Abstract Full Text PDF PubMed Google Scholar, 16Romanik E.A. O'Connor C.M. J. Biol. Chem. 1989; 264: 14050-14056Abstract Full Text PDF PubMed Google Scholar) and because these large cells are easily injected with macromolecules. For the present studies, we constructed an epitope-tagged CaM (17Szymanska G. O'Connor M.B. O'Connor C.M. Anal. Biochem. 1997; 252: 96-105Crossref PubMed Scopus (13) Google Scholar) as a model substrate, enabling us to follow its fate after microinjection into oocytes. CaM was chosen for several reasons. CaM acts as a physiological methyl acceptor in several cell types, including both the human erythrocyte (18Brunauer L.S. Clarke S. Biochem. J. 1986; 236: 811-820Crossref PubMed Scopus (16) Google Scholar, 19Runte L. Jürgensmeier C.U. Söling H.D. FEBS Lett. 1982; 147: 125-130Crossref PubMed Scopus (16) Google Scholar) and theXenopus oocyte (20Desrosiers R.R. Romanik E.A. O'Connor C.M. J. Biol. Chem. 1990; 265: 21368-21374Abstract Full Text PDF PubMed Google Scholar). Careful measurement of the methylation stoichiometry in the two cells has revealed that about 0.02–0.03% of the CaM is carboxyl methylated at steady state, reflecting the low abundance of isoaspartyl residues generally in proteins. Peptide mapping of erythrocyte CaM (21Ota I.M. Clarke S. Arch. Biochem. Biophys. 1990; 279: 320-327Crossref PubMed Scopus (18) Google Scholar) indicated that physiological methylation occurs at isoaspartyl residues in the central helix and calcium-binding sites as well as near the N terminus. Prolonged incubation of native CaM at the near physiological conditions of pH 7.4 and 37 °C for 2 weeks in vitro produces isoaspartyl residues at these same sites (22Ota I.M. Clarke S. Biochemistry. 1989; 28: 4020-4027Crossref PubMed Scopus (38) Google Scholar, 23Potter S.M. Henzel W.J. Aswad D.W. Protein Sci. 1993; 2: 1648-1663Crossref PubMed Scopus (69) Google Scholar), indicating that it is possible to approximate the naturally occurring age-related changes in CaM in the test tube. We have used the same protocols to produce isomerized variants of an epitope-tagged CaM for microinjection into oocytes. The presence of a hemagglutinin (HA) epitope is advantageous in allowing the injected substrate to be detected by immunochemical methods (17Szymanska G. O'Connor M.B. O'Connor C.M. Anal. Biochem. 1997; 252: 96-105Crossref PubMed Scopus (13) Google Scholar). This experimental design has allowed us to evaluate potential roles of PIMT-catalyzed methylation in cellular protein metabolism. Our results show that the presence of isoaspartyl residues severely compromises the stability of CaM in oocyte cytoplasm. PIMT-catalyzed methylation of damaged HA-CaM prior to microinjection increases its stability, while inhibition of the endogenous PIMT activity decreases the stability of injected HA-CaM. These results are most consistent with the involvement of PIMT in the structural repair of damaged proteins in cells and do not support a role for PIMT in targeting damaged proteins for proteolysis. S-Adenosyl-l-[methyl-3H]methionine (AdoMet; 11.2 Ci/mmol) was purchased from NEN Life Science Products. Nonradioactive AdoMet, sinefungin, and reagent grade chemicals were purchased from Sigma. Ecoscint A scintillation fluid was purchased from National Diagnostics (Atlanta, GA). Electrophoresis chemicals were purchased from Bio-Rad. Phenyl-Sepharose was purchased from Amersham Pharmacia Biotech. The mouse monoclonal antibody HA.11, which recognizes the HA epitope YPYDVPDYA, was purchased from Babco (Berkeley, CA). Horseradish peroxidase-conjugated anti-mouse IgG and Lumi-Glo chemiluminescent substrates were purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD). Nitrocellulose Enhance-100 membranes were purchased from American Bioanalytical (Natick, MA). Sexually mature Xenopus females were obtained fromXenopus Express (Homosassa, FL). Epitope-tagged CaM containing the HA epitope YPYDVPDYA at its N terminus (17Szymanska G. O'Connor M.B. O'Connor C.M. Anal. Biochem. 1997; 252: 96-105Crossref PubMed Scopus (13) Google Scholar) was prepared from E. coli N-4830 cells containing a temperature-sensitive cI repressor protein. To induce recombinant HA-CaM synthesis, bacterial cultures were grown overnight at the permissive temperature of 30 °C, and the cultures were then diluted to an A 600 of 0.4–0.5 with medium prewarmed to the nonpermissive temperature of 41 °C to induce HA-CaM synthesis. After growth at 41 °C for 2 h, bacteria were pelleted by centrifugation for 5 min at 8000 × g. The cell pellet was resuspended in 0.02 volumes of ice-cold lysis buffer containing 10 mm Tris, 1 mm EDTA, 5 mm NaCl, 0.5 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 0.25 mg/ml lysozyme. HA-CaM was purified from the cell lysate through the phenyl-Sepharose step essentially as described (24Putkey J.A. Slaughter G.R. Means A.R. J. Biol. Chem. 1985; 260: 4704-4712Abstract Full Text PDF PubMed Google Scholar). Fractions eluting from the phenyl-Sepharose column with EDTA were applied to an additional phenyl-Sepharose (1 ml of packed resin/ml of pooled fractions) column equilibrated in 50 mm Tris, pH 7.4, 5 mmCaCl2, 500 mm NaCl. The column was washed with 5 volumes of starting buffer, after which HA-CaM was eluted with the buffer containing 1 mm EDTA. Deamidated variants of HA-CaM were prepared by incubating solutions of 100 μm HA-CaM in 50 mm HEPES, 1 mm EDTA, pH 7.4, at 37 °C for 2 weeks (9Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar). Control HA-CaM was incubated in the same buffer containing 1 mm CaCl2 in the place of EDTA. Nondenaturing gel electrophoresis was performed on 15% gels using a discontinuous buffer system containing 2 mm EDTA as described (9Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar). Denaturing SDS-PAGE on 15% gels was carried according to the protocol described by Laemmli and Favre (25Laemmli U.K. Favre M. J. Mol. Biol. 1973; 80: 575-599Crossref PubMed Scopus (3025) Google Scholar). CaM binding proteins in the oocyte extract were detected using a modified CaM overlay procedure (17Szymanska G. O'Connor M.B. O'Connor C.M. Anal. Biochem. 1997; 252: 96-105Crossref PubMed Scopus (13) Google Scholar, 26Slaughter G.R. Means A.R. Methods Enzymol. 1987; 139: 133-144Google Scholar) in the presence of either 1 mm CaCl2 or 5 mm EGTA. Western blotting was performed using a primary antibody directed against the HA epitope and using horseradish peroxidase-conjugated anti-mouse IgG as a secondary antibody, as described previously (17Szymanska G. O'Connor M.B. O'Connor C.M. Anal. Biochem. 1997; 252: 96-105Crossref PubMed Scopus (13) Google Scholar). Bound antibody was detected using the Lumi-Glo chemiluminescent detection system, and the intensity of the bands was determined by densitometry employing a scanning laser densitometer (Molecular Dynamics, Inc., Sunnyvale, CA). Quantitation of relative band densities was performed using the ImageQuant software program. Experimental procedures were adjusted so that all data fell within the linear response range of the chemiluminescent detection system. The amounts of HA-CaM recovered in the oocyte extract were estimated from a standard curve of known amounts of HA-CaM. The concentration of HA-CaM in the standard sample was established by amino acid analysis. Protein samples were separated under nondenaturing conditions as described above and electrophoretically transferred to nitrocellulose membranes, which were stained briefly with 0.1% Amido Black in 45% methanol, 2% acetic acid to visualize the proteins. Segments containing the proteins of interest were excised from the blot and cut into 1 × 1-mm pieces, which were immersed in 20 μl of digestion buffer consisting of 1% octylglucopyranoside, 10% acetonitrile, and 100 mmammonium bicarbonate. An 0.5-μl aliquot of trypsin (0.25 μg/ml) was added, and the digest was incubated overnight at 37 °C. Samples were prepared for MALDI-TOF analysis by mixing a 0.3-μl aliquot of the digestion mixture with 0.5 μl of matrix (10 mg/ml α-cyano-4-hydroxycinnamic acid, 0.1% trifluoroacetic acid in 50% acetonitrile) and allowing the mixture to air dry on the sample grid. MALDI-TOF analysis was performed on a PerSeptive Biosystems Linear Voyager Biospectrometry workstation using external calibrants of angiotensin, MH+ of 1297.5 Da and ACTH (18Brunauer L.S. Clarke S. Biochem. J. 1986; 236: 811-820Crossref PubMed Scopus (16) Google Scholar, 19Runte L. Jürgensmeier C.U. Söling H.D. FEBS Lett. 1982; 147: 125-130Crossref PubMed Scopus (16) Google Scholar, 20Desrosiers R.R. Romanik E.A. O'Connor C.M. J. Biol. Chem. 1990; 265: 21368-21374Abstract Full Text PDF PubMed Google Scholar, 21Ota I.M. Clarke S. Arch. Biochem. Biophys. 1990; 279: 320-327Crossref PubMed Scopus (18) Google Scholar, 22Ota I.M. Clarke S. Biochemistry. 1989; 28: 4020-4027Crossref PubMed Scopus (38) Google Scholar, 23Potter S.M. Henzel W.J. Aswad D.W. Protein Sci. 1993; 2: 1648-1663Crossref PubMed Scopus (69) Google Scholar, 24Putkey J.A. Slaughter G.R. Means A.R. J. Biol. Chem. 1985; 260: 4704-4712Abstract Full Text PDF PubMed Google Scholar, 25Laemmli U.K. Favre M. J. Mol. Biol. 1973; 80: 575-599Crossref PubMed Scopus (3025) Google Scholar, 26Slaughter G.R. Means A.R. Methods Enzymol. 1987; 139: 133-144Google Scholar, 27Wallace R.A. Jared D.W. Dumont J.N. Sega M.W. J. Exp. Zool. 1973; 184: 321-334Crossref PubMed Scopus (366) Google Scholar, 28O'Connor C.M. Germain B.J. J. Biol. Chem. 1987; 262: 10404-10411Abstract Full Text PDF PubMed Google Scholar, 29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar, 30Aswad D.W. Deight E.A. J. Neurochem. 1983; 41: 1702-1709Crossref PubMed Scopus (26) Google Scholar, 31Johnson B.A. Shirokawa J.M. Aswad D.W. Arch. Biochem. Biophys. 1989; 268: 276-286Crossref PubMed Scopus (53) Google Scholar, 32Klee C.B. Vanaman T.C. Adv. Protein Chem. 1982; 35: 213-321Crossref PubMed Scopus (737) Google Scholar, 33Walsh M. Stevens F.C. Kuznicki J. Drabikowski W. J. Biol. Chem. 1977; 252: 7440-7443Abstract Full Text PDF PubMed Google Scholar, 34Johnson B.A. Shirokawa J.M. Hancock W.S. Spellman M.W. Basa L.J. Aswad D.W. J. Biol. Chem. 1989; 264: 14262-14271Abstract Full Text PDF PubMed Google Scholar, 35Ota I.M. Clarke S. J. Biol. Chem. 1989; 264: 54-60Abstract Full Text PDF PubMed Google Scholar, 36Murtaugh T.J. Wright L.S. Siegel F.L. J. Neurochem. 1986; 47: 164-172Crossref PubMed Scopus (19) Google Scholar, 37Bischoff R. Kolbe H.V.Y. J. Chromatogr. 1994; 662: 261-278Crossref Scopus (88) Google Scholar, 38Liu D.T. Trends Biotechnol. 1992; 10: 364-369Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 39Gregori L. Marriott D. Putkey J.A. Means A.R. Chau V. J. Biol. Chem. 1987; 262: 2562-2567Abstract Full Text PDF PubMed Google Scholar), MH+ of 2466.7. Spectra were acquired in positive ion mode using an accelerating voltage of 30 kV and a laser power close to threshold. Oocytes were manually dissected from ovaries and incubated in OR-2 saline (27Wallace R.A. Jared D.W. Dumont J.N. Sega M.W. J. Exp. Zool. 1973; 184: 321-334Crossref PubMed Scopus (366) Google Scholar) as described (28O'Connor C.M. Germain B.J. J. Biol. Chem. 1987; 262: 10404-10411Abstract Full Text PDF PubMed Google Scholar). Oocytes were injected with 46 nl of solutions containing various concentrations of HA-CaM as indicated in the figure legends. In some experiments, oocytes were injected with 46 nl of 2.5 mm sinefungin in 10 mm sodium phosphate, pH 6.8, 30 min prior to injection with HA-CaM. Microinjected oocytes were incubated in OR-2 solution at 20 °C in groups of 5 or 10 for various lengths of time. The cells were routinely observed with a microscope to check for damaged oocytes, which were discarded. At the end of each incubation period, groups of oocytes were washed three times in homogenization medium containing 0.25 m sucrose, 1 mm EDTA, 10 mm sodium phosphate, pH 6.9, and then rapidly frozen in 10 volumes of the same medium. Oocytes were stored in this medium at −80 °C until used for biochemical analysis. For analysis, frozen oocytes were rapidly thawed and homogenized by trituration through a micropipet tip. The soluble protein fraction was prepared by centrifuging the homogenate at 100,000 × gfor 20 min. The protein concentration in the cytosolic fraction was determined by the method of Bradford (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar), using bovine serum albumin as the standard. The acidic isozyme of PIMT was purified from calf brain as described (30Aswad D.W. Deight E.A. J. Neurochem. 1983; 41: 1702-1709Crossref PubMed Scopus (26) Google Scholar). To measure methyl-accepting activities, CaM and HA-CaM samples were incubated with 22 nm PIMT, 10 μm S-adenosyl-l-[methyl-3H]methionine (adjusted to a final specific activity of 1 Ci/mmol with nonradioactive AdoMet) in 25 mm MES, pH 6.5, for 2 h at 37 °C in a final volume of 25 μl. Protein carboxyl [3H]methyl esters were quantified as trichloroacetic acid-precipitable material converted to [3H]methanol by base treatment, as described previously (16Romanik E.A. O'Connor C.M. J. Biol. Chem. 1989; 264: 14050-14056Abstract Full Text PDF PubMed Google Scholar). Each assay was done in triplicate and included a negative control lacking substrate, representing the assay background. Carboxyl methylated HA-CaM was prepared for microinjection using the same procedures, except that radioactivity was omitted from the mixture. Previous experiments have shown that the addition of the HA epitope to the N terminus of CaM has no effect on the enzymatic activity of CaM, suggesting that HA-CaM could be used to model CaM functions (17Szymanska G. O'Connor M.B. O'Connor C.M. Anal. Biochem. 1997; 252: 96-105Crossref PubMed Scopus (13) Google Scholar). We have been interested in using CaM as a model substrate for PIMT because of the ease with which CaM can be converted into isoaspartyl-containing variants under the physiologically relevant conditions of a 2-week incubation at pH 7.4 and 37 °C in the presence of EGTA (9Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar, 22Ota I.M. Clarke S. Biochemistry. 1989; 28: 4020-4027Crossref PubMed Scopus (38) Google Scholar, 31Johnson B.A. Shirokawa J.M. Aswad D.W. Arch. Biochem. Biophys. 1989; 268: 276-286Crossref PubMed Scopus (53) Google Scholar). Following such incubations, one detects new electrophoretic variants that migrate more slowly on nondenaturing gels than native CaM due to the spontaneous generation of methyl-accepting isoaspartyl residues at several sites in the protein, including the calcium-binding sites (9Johnson B.A. Langmack E.L. Aswad D.W. J. Biol. Chem. 1987; 262: 12283-12287Abstract Full Text PDF PubMed Google Scholar, 20Desrosiers R.R. Romanik E.A. O'Connor C.M. J. Biol. Chem. 1990; 265: 21368-21374Abstract Full Text PDF PubMed Google Scholar). In order for HA-CaM to effectively serve as a model for carboxyl methylation, it was necessary to demonstrate that the HA epitope did not adversely affect the spontaneous deamidation of CaM to these methyl-accepting forms. In the experiment shown in Fig. 1, we have used the same nondenaturing gel system to characterize CaM and HA-CaM variants produced during incubation of the samples for 14 days at pH 7.4 and 37 °C in the presence of either 1 mm CaCl2 (lanes 1 and 3) or 1 mm EDTA (lanes 2 and 4). In both cases, incubation in the presence of EDTA generates electrophoretically distinct forms that migrate more slowly than the native forms. Essentially identical results are obtained with the CaM and HA-CaM samples, except that the HA-tagged forms migrate more slowly than their untagged counterparts. All of the variants generated by prolonged incubation of HA-CaM are readily detected using a monoclonal antibody to the HA epitope on immunoblots (Fig. 1, right panel). In order for “aged” HA-CaM (Fig. 1) to serve as a model substrate for PIMT-dependent metabolism in cells, it was important to establish that the 2-week incubation did not produce changes in amino acids other than isoaspartyl generation. It was also important to eliminate the possibility that some of the new electrophoretic variants detected in Fig. 1 represented proteolytic fragments of CaM or HA-CaM, particularly in light of the heightened susceptibility of CaM to proteolytic cleavage in the presence of EGTA (32Klee C.B. Vanaman T.C. Adv. Protein Chem. 1982; 35: 213-321Crossref PubMed Scopus (737) Google Scholar, 33Walsh M. Stevens F.C. Kuznicki J. Drabikowski W. J. Biol. Chem. 1977; 252: 7440-7443Abstract Full Text PDF PubMed Google Scholar). Tryptic peptides were therefore prepared from the major variant in each sample in Fig. 1, and the fragments were analyzed using MALDI-TOF mass spectrometry. The mass spectrum for the peptide mixture obtained from native CaM identifies that protein as authentic CaM. All of the masses detected in Fig. 2 A correspond to the singly charged ions of known tryptic fragments, as indicated. In addition, two peptides, corresponding to residues 127–148 (MH+ = 2493) and residues 38–74 (MH+ = 4071), are associated with oxidized methionyl variants 16 or 32 mass units larger than the parent peptide, in good correspondence to the number of methionyl residues in the peptide. These oxidized methionyl variants are characteristically generated during the MALDI-TOF analyses. The addition of the HA epitope to CaM produces a set of tryptic fragments (Fig. 2 B) consistent with the presence of the sequence AYPYDVPDYAM that was added to the N terminus of CaM during the construction of HA-CaM (17Szymanska G. O'Connor M.B. O'Connor C.M. Anal. Biochem. 1997; 252: 96-105Crossref PubMed Scopus (13) Google Scholar). The ions MH+ = 1523, MH+ = 2461, and MH+ = 3350, corresponding to residues 1–13, 1–24, and 1–30 of native CaM, are missing from the HA-CaM samples, to be replaced with ions MH+ = 2810, MH+ = 3746, and MH+ = 4632. The mass spectra obtained from tryptic digests of deamidated CaM and deamidated HA-CaM are largely similar to those obtained with their respective nondeamidated counterparts, with two exceptions. Newly created tryptic ions MH+ = 1598 and MH+ = 3822 (indicated withasterisks in Fig. 2, C and D), corresponding to residues 78–90 and 116–148 of CaM, are detected in both deamidated CaM (Fig. 2 A) and deamidated HA-CaM (Fig. 2 D). These fragments are consistent with a lack of cleavage after Arg86 and Arg126, suggesting that deamidation reduces susceptibility to tryptic cleavage at these two sites. Overall, the incubation used to produce deamidated CaM does not alter the masses of any identified ions, suggesting that changes are limited to aspartyl isomerization and asparaginyl deamidation (21Ota I.M. Clarke S. Arch. Biochem. Biophys. 1990; 279: 320-327Crossref PubMed Scopus (18) Google Scholar, 34Johnson B.A. Shirokawa J.M. Hancock W.S. Spellman M.W. Basa L.J. Aswad D.W. J. Biol. Chem. 1989; 264: 14262-14271Abstract Full Text PDF PubMed Google Scholar,35Ota I.M. Clarke S. J. Biol. Chem. 1989; 264: 54-60Abstract Full Text PDF PubMed Google Scholar). Unfortunately, the resolution of MALDI-TOF analysis is not sufficiently sensitive to confirm the change of 1 mass unit that would be expected from the conversion of an asparaginyl residue to an aspartyl residue. Significantly, ions representing the intact N-terminal and C-terminal peptides of CaM can be identified in all samples, confirming that proteolytic digestion is not responsible for the altered electrophoretic variants generated during the 2-week incubation of CaM used to produce deamidated CaM. This is particularly important in light of a previous report that a brain carboxypeptidase produces a shortened CaM with enhanced methyl-accepting activity (36Murtaugh T.J. Wright L.S. Siegel F.L. J. Neurochem. 1986; 47: 164-172Crossref PubMed Scopus (19) Google Scholar). Because of its unique specificity for protein d-aspartyl andl-isoaspartyl residues, the PIMT has proven to be a useful tool for detecting isoaspartate in peptides and proteins (37Bischoff R. Kolbe H.V.Y. J. Chromatogr. 1994; 662: 261-278Crossref Scopus (88) Google Scholar, 38Liu D.T. Trends Biotechnol. 1992; 10: 364-369Abstract Full Text PDF PubMed Scopus (65) Google Scholar). To determine if the isoaspartyl content of HA-CaM is increased by prolonged incubation in the absence of calcium, we compared the specific methyl-accepting activities of the CaM and HA-CaM produced during a 2-week incubation at 37 °" @default.
• W2029303304 created "2016-06-24" @default.
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• W2029303304 creator A5001819713 @default.
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• W2029303304 date "1998-10-01" @default.
• W2029303304 modified "2023-10-02" @default.
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